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The Drosophila tumor suppressor gene lethal(2)giant larvae is required for the emission of the Decapentaplegic signal

¹ Laboratoire de Génétique et de Physiologie du Développement, UMR 6545 CNRS-Université, IBDM CNRS-INSERM-Université de la Méditerranée, Campus de Luminy, 13288 Marseille cedex 09, France
² Institut de Génétique Humaine, UPR 1142, 141 rue de la Cardonille, 34396 Montpellier cedex 5, France
³ Developmental Immunology-MGH, Jackson Building 14, GRJ 1402, 55 Fruit Street, Boston, MA, 02114, USA

View larger version (144K): [in a new window]   Fig. 1. Distribution of NMHC and membrane markers in lglts3 mutant embryos at dorsal closure. Whole-mount embryos were stained at the beginning of dorsal closure as follows and observed under a confocal microscope: with anti-NMHC (A-D), with anti-Fasciclin III (FasIII), which labels the lateral membrane domain of epithelial cells (E,F), with anti-Phosphotyrosine (anti-PY), a marker of the most apical region of the lateral membrane (G,H), and with anti-Discs-large (Dlg), a marker of septate junctions (I, J). (A,B,C,E,G,I) Wild-type embryos; (D,F,H,J) lglts3 mutant embryos at 29°C. Inserts (G-J) show z-views of dorsal epidermis cells. (A-C) NMHC is strongly expressed in the LE cells, where it accumulates at the onset of dorsal closure. (D) In mutant embryos, LE cells do not change their shape and fail to accumulate NMHC (compare with C, a wild-type embryo (slightly younger than those in A,B) that has not yet changed its LE cells shape). (E) Fas III and (I) Dlg are absent from the membranes facing the amnioserosa in the LE cells during dorsal closure, while in lglts3 embryos, FasIII (F) and Dlg (J) are evenly distributed on the whole surface of the cell membranes. (G) In LE cells, PY is detected in the membrane facing the amnioserosa, as well as in the basolateral membranes in contrast to the situation prevailing in mutant embryos (H). In other ectodermal cells, epithelial cell polarity is maintained in lglts3 mutant embryos and cell junctions are correctly positioned (inserts in G-I). Arrowheads indicate the LE cells.

View larger version (131K): [in a new window]   Fig. 2. Downregulation of the expression of dpp targets in lglts3 mutant embryos. (A,D,F,H,J) Wild-type embryos; (C,E,G,I,K) lglts3 homozygous embryos reared at 29°C; (B) tkv7 homozygous embryo. (A-C) In situ hybridizations of whole-mount embryos probed with a DIG-labeled zipper cDNA. The views are focused on one of the two LE cells rows (arrowheads) where zip transcript accumulates in a wild-type embryo at the onset of dorsal closure (A) but fails to accumulate either in a tkv7 homozygous embryo (B) or in a lglts3 homozygous embryo at 29°C (C). as, amnioserosa; ec, lateral ectoderm; h, head. (D,E) In situ hybridizations of whole-mount embryos probed with a DIG-labeled dpp cDNA. (D) In a stage 13 wild-type embryo, dpp is expressed in the LE cells (arrowhead), as well as in subsets of other cells such as those in the visceral mesoderm. (E) A lglts3 mutant embryo at the same stage expresses dpp in the LE cells (arrowhead), as does the wild-type embryo. Note the lower expression of dpp in the PS7 visceral mesoderm. (F,G) In situ hybridizations of whole-mount embryos probed with a DIG-labeled lab cDNA. lab expression was no longer detected in a lglts3 mutant embryo (arrowhead). The most extreme phenotype, with almost complete absence of lab expression, is shown here. Persistence of a labeling in the intercalary segment should be noted in mutant embryos. (H,I) Tin is expressed in the cardial cells in wild-type embryos and absent from most of the cardial cells in mutant embryos (arrowheads). (J,K) The same situation prevails in the case of Eve expression. The anti-Eve antibody labels a subset of pericardial cells and of dorsal muscles precursors. For all embryos shown, anterior is leftwards and the dorsal side is in focus. The genotypes are mentioned in the lower left and the probe or the antibody used in the lower right of each panel.

View larger version (128K): [in a new window]   Fig. 3. The dorsoventral patterning of the embryonic ectoderm mediated by the dpp signaling is affected in lglts3 mutant embryos. (A-D) Cuticles of lglts3 homozygous embryos reared at 29°C. (A) Absence of dorsal closure producing a large hole in the dorsal ectoderm is visible in 58% of lglts3 mutant embryos. (B) Shows the twisted phenotype and a lack of involution of the head. (C) The absence of cephalopharyngeal skeleton (arrow) and the lack of externalization (or the absence) of the Filzkörpers (arrowhead) were the most frequently encountered phenotypes. (D) Mutant cuticle showing a rare phenotype consisting in a partial lack of ventral cuticle (arrowhead). Cuticle preparations of wild-type (E,G,I) or lglts3 mutant (F,H,J) embryos in which have been expressed Dpp (E,F,I,J) or constitutively activated Tkv (G,H) under the control of prd (E-H) or en (I,J) regulatory elements. The extent of dorsalization was assessed by the reduction of the number of ventral cuticles (arrowheads). Dorsalization by ectopic expression of Dpp in wild-type embryos (E,I) is more pronounced than that induced by TkvQ253D in wild-type embryos (G; data not shown) probably because Dpp can diffuse some distance away from its source of production. As prd is a pair-ruled gene, dorsalization is observed in only one of two segments in (E,H). Cuticle in F is very similar to that in wild-type, indicating that lgl is required for dpp mediated dorsalization (compare E with F). More than 80% of mutant cuticles were phenotypically wild-type whereas in control embryos 100% of them were dorsalized. By contrast, the extent of dorsalization is roughly identical in G,H, indicating that TkvQ253D can rescue lgl function in this process. (I,J) When dpp is induced in en-expressing cells, the ventral denticles of all the segments can potentially be affected owing to the expression of en in the epidermal cells that gives rise to the anteriormost row of denticle belts. Dorsalization was also more effective in wild-type (I) than in lglts3 embryos (J). In all panels, cuticles are oriented with the anterior towards the left. Arrowheads point to wild-type (F,J) or phenotypically mutant (E,G-I) denticle belts.

View larger version (64K): [in a new window]   Fig. 4. Expression of Spalt, a dpp target, in lgl4 imaginal wing discs. In all panels (except E,F), Spalt (Sal) expression was revealed using an anti-Spalt antibody (in red). (A,B) The expression of Sal in the center of the presumptive wing blade (arrowhead) is abolished in early third instar mutant larva. The dpp-independent expression of Sal in the notum (arrow) is not affected by lgl loss of function. (C,D) dpp expression, revealed by in situ hybridization with a DIG-labeled dpp probe (in green) is normally initiated in a lgl4 mutant disc from early third instar larva. (E,F) En face optical sections of imaginal discs. Epithelial cell polarity probed with anti-Dlg (green) and anti-Arm (red) antibodies. In spite of a disorganization of the epithelial folds in lgl4 mutant discs (F), Dlg and Arm are normally located at the plasma membranes. (E',F') z-views of the epithelial layers at positions marked by an arrow in (E,F). As in the wild type, Arm is located in the mutant to the apicalmost region of the lateral membrane, above the septate junctions labeled with Dlg. (G-R) All discs are lgl4 mutants. (I,L,O,R) are merged views of the two separate views on their left. (G-I) en-Gal4 driven expression of a constitutively activated form of Tkv in the posterior compartment of a lgl4 mutant disc induces the expression of Sal in the posterior compartment, marked with anti-En antibody. The star indicates a trachea. (J-L) Under the same conditions as in (G-I), expression of dpp in en-expressing cells is unable to induce Sal expression in the posterior compartment (arrowhead). (M-O) Sal expression is restored in the presumptive wing blade (arrowhead) of an lgl4 mutant wing disc by expression of lgl under the control of dpp-Gal4. The dpp domain of expression was assessed by UAS-GFP (green). (P-R) expression of lgl in the posterior domain of a lgl4 mutant disc cannot rescue Sal expression in the presumptive wing blade (arrowhead). The arrows in M-R point to dpp-independent Sal expression.

View larger version (98K): [in a new window]   Fig. 5. lgl function in the eye imaginal disc. (A,B,D) Anti-Sal staining, which labels a small number of photoreceptors in every differentiated ommatidia. (C) In situ hybridization with a DIG-labeled dpp probe. At late third instar larval stage, lgl mutant eye discs are smaller than in the wild type (compare the respective sizes of the eye disc with the antenna disc in A,B, for example). (D) The differentiation of ommatidia is almost completely restored when lgl is expressed in lgl mutant discs under the control of dpp-Gal4.

View larger version (82K): [in a new window]   Fig. 6. Non-autonomous behavior of lgl4 mutant clones with respect to dpp signaling in wing imaginal discs. Homozygous clones for lgl4 have been produced by the FLP-FRT system in a wild-type (A-D) or in a Minute background (E-H). Mitotic recombinations have been induced during the second larval stage. Mutant clones were visualized by the absence of the arm-lacZ marker (green in A,E). The activity of the dpp signaling pathway was assessed by the expression of Spalt (red in B,F). The position of the A/P boundary was assigned by the expression of Patched (blue in C,G). (D,H) Merged images. Filled arrowheads point to mutant clones. The large arrows in A,D show an example of a twin clone lying just anteriorly to the A/P boundary. a, anterior; d, dorsal. The general structure of the wing discs containing numerous large mutant clones was repeatedly deeply affected.

View larger version (91K): [in a new window]   Fig. 7. Mosaic analysis of lgl4 wings. (A) Example of lgl4 clones occupying dorsal and ventral wing blade surfaces and causing loss of veins. (B) Schematic representation of ventral (unbroken lines) and dorsal (broken lines) clones in (A) of homozygous lgl4 (-/-) and twin wild-type (+/+) clones. (C) Example of a lgl4 clone (broken line) encompassing the posterior cross-vein that differentiates despite the presence of a large clone in the ventral surface. (D) Example of ectopic vein material within a lgl4 mutant clone.